{"doi":"10.1016/j.xpro.2024.103221","title":"Protocol for establishing inducible CRISPR interference system for multiple-gene silencing in human pluripotent stem cells","abstract":"Inducible loss-of-function strategies are crucial for understanding gene function. However, creating inducible, multiple-gene knockout models is challenging and time-consuming. Here, we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs, designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid, and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones. For complete details on the use and execution of this protocol, please refer to Matsui et al. 1 • Dox-inducible CRISPRi system to silence multiple genes concurrently • Instructions for generating CRISPRi hPSCs transduced with four sgRNAs • FOXA1/A2/A3 -CRISPRi system represses expression of all three FOXA genes by 95% Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Inducible loss-of-function strategies are crucial for understanding gene function. However, creating inducible, multiple-gene knockout models is challenging and time-consuming. Here, we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs, designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid, and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones.","journal":"STAR Protocols","year":2024,"id":468728,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9577,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1012701,"name":"Joseph R Shiley","orcid":"0000-0001-8453-1738","position":1,"is_corresponding":false},{"id":932785,"name":"Marissa Granitto","orcid":"0000-0002-1758-9881","position":2,"is_corresponding":false},{"id":1207852,"name":"Katie Ludwig","orcid":null,"position":3,"is_corresponding":false},{"id":1012700,"name":"Morgan Buckley","orcid":"0009-0008-2015-7730","position":4,"is_corresponding":false},{"id":1207853,"name":"Sandra Koigi","orcid":null,"position":5,"is_corresponding":false},{"id":1302214,"name":"Gerardo Mirizio","orcid":null,"position":6,"is_corresponding":false},{"id":87797,"name":"Yueh-Chiang Hu","orcid":"0000-0002-7992-0525","position":7,"is_corresponding":false},{"id":301666,"name":"Christopher N. Mayhew","orcid":"0000-0002-7408-1726","position":8,"is_corresponding":false},{"id":289719,"name":"Makiko Iwafuchi","orcid":"0000-0003-1291-6246","position":9,"is_corresponding":false},{"id":1012699,"name":"Satoshi� Matsui","orcid":"0000-0002-6315-4109","position":0,"is_corresponding":true}],"reference_count":24,"raw_metadata":null,"created_at":"2026-07-19T02:05:23.500722Z","pmid":"39083383","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}